Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of infectious neurodegenerative disorders affecting humans and animals. Although rare diseases, the fact that Bovine Spongiform Encephalopathy (BSE) is present in North America and the continuous spread of Chronic Wasting Disease (CWD), have augmented concerns about a possible problem for human health. This research project builds around three important recent findings from my laboratory: 1.) The discovery of unusual BSE cases in the U.S. cattle population. 2.) The discovery of the first and so far only case of a genetic form (E211K) of an animal TSE in the 2006 U.S. BSE case. 3.) The generation of PrP knock-out cattle that develop healthy up to an adult stage. These three findings provide us with unique tools to investigate several aspects of BSE pathogenesis. The major goal of this project is to generate and characterize mutant knock-in and knock-out cows and assess disease transmission, susceptibility and species barrier in diverse forms of BSE using transgenic mice and in vitro conversion experiments. In specific aim 1 we plan to characterize PrP knock-out cattle in more detail and generate and characterize PrP knock-in cattle expressing the mutation E211K in the PrP null background. In specific aim 2 we will study the susceptibility of PrP bovinized mice expressing the E211K mutation to various forms of BSE. Specific aim 3 will evaluate transmission of E211K bovine prions and other BSE isolates to transgenic mice expressing PrP from various animal species, including sheep, deer, and human as well as wild type mice and hamsters. In specific aim 4 we plan to evaluate the differential susceptibility of PrPc from various species (human, sheep, cattle and deer) to be converted in vitro by PrPSc derived from various forms of BSE. The findings obtained in this project will provide a substantial advance on our understanding of BSE pathogenesis, transmission and species barrier and will likely have great impact in public health and the regulatory measures to prevent further spreading of this disease.